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Neoclerodane diterpenoids from Teucrium polium subsp. polium and their antifeedant activity
Biochemical Systematics and Ecology 31 (2003) 1051–1056 www.elsevier.com/locate/biochemsyseco
Neoclerodane diterpenoids from Teucrium polium subsp. polium and their antifeedant activity Maurizio Bruno a,∗, Antonella M. Maggio a, Franco Piozzi a, Suzette Puech b, Sergio Rosselli a, Monique S.J. Simmonds c a
Dipartimento Chimica Organica, Universita` di Palermo, Viale delle Scienze — Parco d’Orleans II, 90128 Palermo, Italy b Institut de Botanique, Universite´ Montpellier II, Montpellier (MPU), France c Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK Received 20 October 2002; accepted 3 January 2003
Keywords: Lamiaceae; Diterpenes; Neoclerodanes; Teucrium; Antifeedant; Spodoptera littoralis
1. Subject and source Neoclerodane diterpenoids have been isolated from plants generically known as “Teucrium polium” and collected from different places of origin: Southern France (Brieskorn and Pfeuffer, 1967), Bulgaria (Malakov et al., 1979, 1982, 1988; Malakov and Papanov, 1983; Papanov and Malakov, 1983; Gacs-Baitz et al., 1987), Moldovia (Popa et al., 1977), Armenia (Galstyan et al., 1992) and Turkey (Bedir et al., 1999). The diterpenoids isolated from these sources differed qualitatively. As far as we know, no previous work has been published on an authenticated sample of T. polium subsp. polium. We report here our investigation on diterpenoids from authenticated material collected in Southern France. Aerial parts of T. polium subsp. polium were collected at Traviargues (AnduzeGard) in October 2001 and identified by Prof. S. Puech. A specimen (voucher no. 2501a S. Puech) is deposited in the herbarium of the Institut de Botanique, Universite´ de Montpellier II.
∗
Corresponding author. Tel.: +39-091-596905; fax: +39-091-596825. E-mail address: [email protected] (M. Bruno).
0305-1978/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0305-1978(03)00042-5
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M. Bruno et al. / Biochemical Systematics and Ecology 31 (2003) 1051–1056
2. Previous work T. polium L. (Lamiaceae) has been the subject of several investigations in the last 30 years, with the purpose of elucidating the structure of neoclerodane diterpenoids occurring in the aerial parts of this and other species of Teucrium (Piozzi, 1981, 1994; Piozzi et al., 1987, 1998). The taxonomy of the section Polium of the genus is complex and is believed to contain 125 taxa (Navarro and El Oualidi, 2000); in the case of T. polium, several subspecies were described. 3. Present study Air dried and finely powdered aerial parts (130 g) were extracted three times with (CH3)2CO (1 l) at room temperature for 1 week. After filtration, the solvent was removed under reduced pressure to yield 11.2 g of material. The residue was chromatographed on a silica gel column, eluting with pet.ether with increasing percentages of AcOEt and collected as 100 ml fractions: 1–10 (pet.ether), 11–18 (pet.ether:AcOEt 9:1), 19–31 (pet.ether:AcOEt 3:1), 32–46 (pet.ether:AcOEt 1:1), 47–52 (pet.ether:AcOEt 2:3), 53–57 (pet.ether:AcOEt 3:7), 58–65 (pet.ether:AcOEt 1:4), 66–73 (AcOEt).
M. Bruno et al. / Biochemical Systematics and Ecology 31 (2003) 1051–1056
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Fractions 47–52 were rechromatographed. A subfraction contained 25 mg of an unresolvable mixture of auropolin (1a), representing about 80% of the total and of a new similar product (MS indicated the occurrence of only one molecular ion at 462 m/z for C24H30O9). The following subfraction yielded 20 mg of pure auropolin (1a). Auropolin was previously found in T. aureum Schreb., formerly T. polium subsp. aureum (Eguren et al., 1981) and in T. capitatum, formerly T. polium subsp. capitatum (Camps et al., 1987). Physical and spectroscopic data were in full agreement with those previously reported for 1a. Fractions 53–57 were rechromatographed giving capitatin (2) (19 mg) as the only component. Capitatin was isolated previously only from T. capitatum, formerly T. polium subsp. capitatum (Marquez et al., 1980). Physical and spectroscopic data were in full agreement with those reported previously for 2. It was not possible to isolate the new product accompanying auropolin in the mixture. Therefore, its structure was elucidated by a careful study of the NMR spectra of the mixture. The 1H and 13C NMR spectra showed the known signals of auropolin, and some additional signals that could be attributed to the second component, for which we propose the structure (3a) of 20-epi-auropolin. In the 1H NMR spectrum, signals occurred for H-20 (broad doublet at 5.38 δ for 3a; sharp doublet at 5.25 δ for 1a), H-7 (singlet at 3.83 δ for 3a; singlet at 4.13 δ for 1a), H-19A (doublet at 5.70 δ for 3a; doublet at 4.98 δ for 1a), H-19B (doublet at 4.99 δ for 3a; doublet at 4.90 δ for 1a) and CH3-17 (singlet at 1.00 δ for 3a; singlet at 1.19 δ for 1a). The most significant differences in the 13C NMR spectrum were observed for C-7 (89.0 δ in 3a; 90.8 δ in 1a), C-9 (50.6 δ in 3a; 53.5 δ in 1a), C-11 (33.5 δ in 3a; 31.5 δ in 1a), C-17 (14.6 δ in 3a; 15.3 δ in 1a) and C-20 (104.8 δ in 3a; 99.5 δ in 1a). These signals agreed with the structure (3a) of 20-epi-auropolin. The configuration at C-20 of compound 3a was suggested to be R, and not S as in auropolin (1a), because of the presence of a long range coupling ( 4J 20,10b ⬍ 0.5 Hz) that has been observed for teucossin B (Alcazar et al., 1992) and montanin H (Malakov et al., 1992), compounds with the same R stereochemistry at C-20 (Table 1). The hypothesis was confirmed by NOE experiment. In fact, irradiation of the Me-17 protons at 1.00 δ caused clear NOE enhancements in the signals of H-20 at 5.38 δ (4.8%), H7β at 3.83 δ (6.5%) and H-8β at 1.97 δ (5.1%), and irradiation of H-7β at 3.83 δ caused NOE enhancement of the signal of Me-17 protons at 1.00 δ (1.3%). These facts unambiguously established that the Me-17 and H-20 protons are on the same side of the plane defined by the C-20/C-7 hemiacetal ring. On the other hand, irradiation of the Me-17 protons of the main compound, auropolin (1a), at 1.19 δ caused NOE enhancements in the signals of H-7β at 4.13 δ (11.3%), H-12 at 5.87d δ (2.9%) and H-8β at 1.97 δ (6.2%) but not of H-20 at 5.25 δ. Finally, all the previous observations were confirmed by the correlation peaks shown in a ROESY spectrum. The spectroscopic conclusions are supported by the fact that oxidation of the mixture with pyridinium bichromate gave only one product, identified with the known lactone 4 (Eguren et al., 1981). In order to try a chromatographic separation of 1a and 3a, we acetylated the mixture, obtaining an unresolvable mixture of the two
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Table 1 NMR data of compounds 1a and 3a (CDCl3). Bruker AMX-600 instrument at 600 MHz resp. 150.8 MHz for the mixture of 1a and 3a and for ROESY and NOE experiments Proton
1a
3a
JH,H (Hz)
C
1a
3a
1α 1β 2α 2β 3α 3β 7β 8β 10β 11 (2H) 12 14 15 16 Me17 18A 18B 19A 19B 20 Ac Ac OH
1.57 dddd
a
a
a
1α,1β = 12.7 1α,2α = 3.4 1α,2β = 12.7 1α,10β = 12.7 1β,10β = 1.4 2α,2β = 13.0 3α,3β = 13.6 7β,8β = 0 8β,17 = 7.0 11,12 = 6.3 14,15 = 1.7 14,16 = 0.7 15,16 = 1.7 18A,18B = 12.7 18A,3α = 2.1 (4J) 19A,19B = 11.4 20,OH = 3.0 20,10βⱕ0.5 (4J)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ac
21.1 t 24.5 t 31.5 t 62.4 s 52.7 s 204.0 s 90.8 d 46.9 d 53.5 s 51.1 d 31.5 t 66.2 d 125.5 s 108.6 d 143.6 d 140.0 d 15.3 q 49.0 t 62.9 t 99.5 d 171.1 s 170.1 s 21.5 q 20.9 q
22.7 t 25.8 t 31.9 t 62.4 s 53.0 s 203.1 s 89.0 d 47.9 d 50.6 s 52.3 d 33.5 t 65.6 d 125.3 s 108.5 d 143.7 d 140.1 d 14.6 q 48.8 t 64.1 t 104.8 d 171.4 s 170.0 s 21.4 q 21.1 q
a
2.18 1.45 2.38 1.04 4.13 1.97 1.96 2.32 5.87 6.41 7.37 7.42 1.19 3.03 2.37 4.98 4.90 5.25 2.07 2.03 3.38
m m m m s q dd d t dd dd m d dd d d d d s s d
a a a a
3.83 s a a
2.32 5.87 6.40 7.38 7.42 1.00 2.96 2.38 5.70 4.99 5.38 2.07 2.02 4.07
d t dd dd m d dd d d d brd s s d
Overlapped signal.
acetyl derivatives 1b and 3b. Pure acetyl-auropolin (1b) was obtained by Ac2Opyridine treatment of pure auropolin (1a) (Eguren et al., 1981). The hypothesis of a dynamic equilibration between the two hemiacetals 1a and 3a could be ruled out. In fact, auropolin (1a) proved to be stable under mild acidic treatment. Moreover, in other Teucrium species, only one of the two possible hemiacetals was detected, but never both together.
4. Chemotaxonomic significance The distribution of neoclerodanes suggests that T. capitatum is taxonomically close to T. polium subsp. polium as both species contain both capitatin and auropolin; however, T. capitatum also contains many other diterpenoids not present in T. polium subsp. polium. The fact that the diterpenoids occurring in all the taxa indicated as T. polium are
M. Bruno et al. / Biochemical Systematics and Ecology 31 (2003) 1051–1056
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distinctly different from those isolated from our authentic T. polium subsp. polium prompts the question of a revision of these identifications.
5. Antifeedant activity A binary choice bioassay using sucrose treated glass-fibre discs (Whatman 2.1 cm diameter) was used to investigate the antifeedant activity of the compounds against final stadium larvae of Spodoptera littoralis (Simmonds et al., 1990) (Table 2). The amounts eaten of the neoclerodane treated (T) and control (C) discs were used to calculate the Feeding Index ((C–T) / (C + T))%. In this index, a positive value indicates an antifeedant. The compounds were tested at a range of concentration from 1 to 1000 ppm to establish whether the compounds gave dose-dependent responses and linear regression was used to calculate the concentration required to give a Feeding Index of 50% (FI50). The Feeding Index at 100 ppm is presented in the results in order to enable comparisons to be made with the activity of other neoclerodanes. The Wilcoxon matched-pairs test was used to analyse the data. A comparison of the antifeedant activity against S. littoralis of the synthetic compound acetyl-auropolin (1b) with the lack of activity of auropolin (1a) shows the importance of the esterification of the hydroxy group at C-20. Compound 4 with a lactone at C-20, although not active at 100 ppm, does elicit antifeedant activity at higher concentration and an FI50 of 230 ppm. Capitatin (2) which lacks a C-7 to C9 bridge showed significant activity at 100 ppm but the activity was not dose-dependent. Previous research on capitatin at 100 ppm showed a Feeding Index (mean (s.e.m.)) of 45 (3.4) and 26 (8.4) against S. littoralis and S. frugiperda, respectively (Simmonds and Blaney, 1992).
Table 2 Effect of compounds 1a, 1b, 2, 4 on the feeding of final stadium larvae of S. littoralis (n = 5–15 per concentration) Compound
1a 1b 2 4
Feeding Indexa at 100 ppm Mean
(SEM)
17.8 57.8∗ 31.0∗ 6.9
7.10 13.64 11.22 19.99
FI50b (ppm)
ndr 90 ndr 230
Feeding Index ((C⫺T) / (C + T))% at 100 ppm (n = 10). Concentration (ppm) required to elicit a 50% Feeding Index: ndr = no linear dose - dependent response. ∗ P ⬍ 0.05 Wilcoxon matched-pairs test. a
b
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Acknowledgements The present work was supported by a grant of the Italian Ministry of University and Research (MIUR). We thank Dr Paul Green for technical assistance with the insects, reared under a Plant Health licence from DEFRA, UK.
References Alcazar, R., de la Torre, M.C., Rodriguez, B., Bruno, M., Piozzi, F., Savona, G., Arnold, N.A., 1992. Phytochemistry 31, 3957. Bedir, E., Tasdemir, D., Calis, I., Zerbe, O., Sticher, O., 1999. Phytochemistry 51, 921. Brieskorn, C.H., Pfeuffer, T., 1967. Chem. Ber. 100, 1998. Camps, F., Coll, J., Dargallo, O., Rius, J., Miravitlles, C., 1987. Phytochemistry 26, 1475. Eguren, L., Perales, A., Fayos, J., Savona, G., Paternostro, M.P., Piozzi, F., Rodriguez, B., 1981. J. Org. Chem. 46, 3364. Gacs-Baitz, E., Papanov, G.Y., Malakov, P.Y., Szilagyi, L., 1987. Phytochemistry 26, 2110. Galstyan, A.M., Shashkov, A.S., Oganesyan, G.B., Mnatsakanian, V.A., Serebryakov, E.P., 1992. Khim. Prirod. Soedin. 28, 503. Malakov, P.Y., Papanov, G.Y., 1983. Phytochemistry 22, 2791. Malakov, P.Y., Papanov, G.Y., Mollov, N.M., 1979. Z. Naturforsch. 34b, 1570. Malakov, P.Y., Papanov, G.Y., Ziesche, J., 1982. Phytochemistry 21, 2597. Malakov, P.Y., Boneva, I.M., Papanov, G.Y., Spassov, S.L., 1988. Phytochemistry 27, 1141. Malakov, P.Y., Papanov, G.Y., Boneva, I.M., 1992. Phytochemistry 31, 4029. Marquez, C., Rabanal, R.M., Valverde, S., Eguren, L., Perales, A., Fayos, J., 1980. Tetrahedron Lett., 5039. Navarro, T., El Oualidi, J., 2000. Flora Mediterr., 349. Papanov, G.Y., Malakov, P.Y., 1983. Phytochemistry 22, 2787. Piozzi, F., 1981. Heterocycles 15, 1489. Piozzi, F., 1994. Heterocycles 37, 603. Piozzi, F., Bruno, M., Rosselli, S., 1998. Heterocycles 48, 2185. Piozzi, F., Savona, G., Rodriguez, B., 1987. Heterocycles 25, 807. Popa, D.P., Fan Tkhuk, A.N., Salei, L.A., 1977. Khim. Prirod. Soedin. 13, 49. Simmonds, M.S.J., Blaney, W.M., 1992. In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew, UK, pp. 375–392. Simmonds, M.S.J., Blaney, W.M., Fellows, L.E., 1990. J. Chem. Ecol. 16, 3167.
Neoclerodane diterpenoids from Teucrium polium subsp. polium and their antifeedant activity Maurizio Bruno a,∗, Antonella M. Maggio a, Franco Piozzi a, Suzette Puech b, Sergio Rosselli a, Monique S.J. Simmonds c a
Dipartimento Chimica Organica, Universita` di Palermo, Viale delle Scienze — Parco d’Orleans II, 90128 Palermo, Italy b Institut de Botanique, Universite´ Montpellier II, Montpellier (MPU), France c Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK Received 20 October 2002; accepted 3 January 2003
Keywords: Lamiaceae; Diterpenes; Neoclerodanes; Teucrium; Antifeedant; Spodoptera littoralis
1. Subject and source Neoclerodane diterpenoids have been isolated from plants generically known as “Teucrium polium” and collected from different places of origin: Southern France (Brieskorn and Pfeuffer, 1967), Bulgaria (Malakov et al., 1979, 1982, 1988; Malakov and Papanov, 1983; Papanov and Malakov, 1983; Gacs-Baitz et al., 1987), Moldovia (Popa et al., 1977), Armenia (Galstyan et al., 1992) and Turkey (Bedir et al., 1999). The diterpenoids isolated from these sources differed qualitatively. As far as we know, no previous work has been published on an authenticated sample of T. polium subsp. polium. We report here our investigation on diterpenoids from authenticated material collected in Southern France. Aerial parts of T. polium subsp. polium were collected at Traviargues (AnduzeGard) in October 2001 and identified by Prof. S. Puech. A specimen (voucher no. 2501a S. Puech) is deposited in the herbarium of the Institut de Botanique, Universite´ de Montpellier II.
∗
Corresponding author. Tel.: +39-091-596905; fax: +39-091-596825. E-mail address: [email protected] (M. Bruno).
0305-1978/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0305-1978(03)00042-5
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M. Bruno et al. / Biochemical Systematics and Ecology 31 (2003) 1051–1056
2. Previous work T. polium L. (Lamiaceae) has been the subject of several investigations in the last 30 years, with the purpose of elucidating the structure of neoclerodane diterpenoids occurring in the aerial parts of this and other species of Teucrium (Piozzi, 1981, 1994; Piozzi et al., 1987, 1998). The taxonomy of the section Polium of the genus is complex and is believed to contain 125 taxa (Navarro and El Oualidi, 2000); in the case of T. polium, several subspecies were described. 3. Present study Air dried and finely powdered aerial parts (130 g) were extracted three times with (CH3)2CO (1 l) at room temperature for 1 week. After filtration, the solvent was removed under reduced pressure to yield 11.2 g of material. The residue was chromatographed on a silica gel column, eluting with pet.ether with increasing percentages of AcOEt and collected as 100 ml fractions: 1–10 (pet.ether), 11–18 (pet.ether:AcOEt 9:1), 19–31 (pet.ether:AcOEt 3:1), 32–46 (pet.ether:AcOEt 1:1), 47–52 (pet.ether:AcOEt 2:3), 53–57 (pet.ether:AcOEt 3:7), 58–65 (pet.ether:AcOEt 1:4), 66–73 (AcOEt).
M. Bruno et al. / Biochemical Systematics and Ecology 31 (2003) 1051–1056
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Fractions 47–52 were rechromatographed. A subfraction contained 25 mg of an unresolvable mixture of auropolin (1a), representing about 80% of the total and of a new similar product (MS indicated the occurrence of only one molecular ion at 462 m/z for C24H30O9). The following subfraction yielded 20 mg of pure auropolin (1a). Auropolin was previously found in T. aureum Schreb., formerly T. polium subsp. aureum (Eguren et al., 1981) and in T. capitatum, formerly T. polium subsp. capitatum (Camps et al., 1987). Physical and spectroscopic data were in full agreement with those previously reported for 1a. Fractions 53–57 were rechromatographed giving capitatin (2) (19 mg) as the only component. Capitatin was isolated previously only from T. capitatum, formerly T. polium subsp. capitatum (Marquez et al., 1980). Physical and spectroscopic data were in full agreement with those reported previously for 2. It was not possible to isolate the new product accompanying auropolin in the mixture. Therefore, its structure was elucidated by a careful study of the NMR spectra of the mixture. The 1H and 13C NMR spectra showed the known signals of auropolin, and some additional signals that could be attributed to the second component, for which we propose the structure (3a) of 20-epi-auropolin. In the 1H NMR spectrum, signals occurred for H-20 (broad doublet at 5.38 δ for 3a; sharp doublet at 5.25 δ for 1a), H-7 (singlet at 3.83 δ for 3a; singlet at 4.13 δ for 1a), H-19A (doublet at 5.70 δ for 3a; doublet at 4.98 δ for 1a), H-19B (doublet at 4.99 δ for 3a; doublet at 4.90 δ for 1a) and CH3-17 (singlet at 1.00 δ for 3a; singlet at 1.19 δ for 1a). The most significant differences in the 13C NMR spectrum were observed for C-7 (89.0 δ in 3a; 90.8 δ in 1a), C-9 (50.6 δ in 3a; 53.5 δ in 1a), C-11 (33.5 δ in 3a; 31.5 δ in 1a), C-17 (14.6 δ in 3a; 15.3 δ in 1a) and C-20 (104.8 δ in 3a; 99.5 δ in 1a). These signals agreed with the structure (3a) of 20-epi-auropolin. The configuration at C-20 of compound 3a was suggested to be R, and not S as in auropolin (1a), because of the presence of a long range coupling ( 4J 20,10b ⬍ 0.5 Hz) that has been observed for teucossin B (Alcazar et al., 1992) and montanin H (Malakov et al., 1992), compounds with the same R stereochemistry at C-20 (Table 1). The hypothesis was confirmed by NOE experiment. In fact, irradiation of the Me-17 protons at 1.00 δ caused clear NOE enhancements in the signals of H-20 at 5.38 δ (4.8%), H7β at 3.83 δ (6.5%) and H-8β at 1.97 δ (5.1%), and irradiation of H-7β at 3.83 δ caused NOE enhancement of the signal of Me-17 protons at 1.00 δ (1.3%). These facts unambiguously established that the Me-17 and H-20 protons are on the same side of the plane defined by the C-20/C-7 hemiacetal ring. On the other hand, irradiation of the Me-17 protons of the main compound, auropolin (1a), at 1.19 δ caused NOE enhancements in the signals of H-7β at 4.13 δ (11.3%), H-12 at 5.87d δ (2.9%) and H-8β at 1.97 δ (6.2%) but not of H-20 at 5.25 δ. Finally, all the previous observations were confirmed by the correlation peaks shown in a ROESY spectrum. The spectroscopic conclusions are supported by the fact that oxidation of the mixture with pyridinium bichromate gave only one product, identified with the known lactone 4 (Eguren et al., 1981). In order to try a chromatographic separation of 1a and 3a, we acetylated the mixture, obtaining an unresolvable mixture of the two
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Table 1 NMR data of compounds 1a and 3a (CDCl3). Bruker AMX-600 instrument at 600 MHz resp. 150.8 MHz for the mixture of 1a and 3a and for ROESY and NOE experiments Proton
1a
3a
JH,H (Hz)
C
1a
3a
1α 1β 2α 2β 3α 3β 7β 8β 10β 11 (2H) 12 14 15 16 Me17 18A 18B 19A 19B 20 Ac Ac OH
1.57 dddd
a
a
a
1α,1β = 12.7 1α,2α = 3.4 1α,2β = 12.7 1α,10β = 12.7 1β,10β = 1.4 2α,2β = 13.0 3α,3β = 13.6 7β,8β = 0 8β,17 = 7.0 11,12 = 6.3 14,15 = 1.7 14,16 = 0.7 15,16 = 1.7 18A,18B = 12.7 18A,3α = 2.1 (4J) 19A,19B = 11.4 20,OH = 3.0 20,10βⱕ0.5 (4J)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ac
21.1 t 24.5 t 31.5 t 62.4 s 52.7 s 204.0 s 90.8 d 46.9 d 53.5 s 51.1 d 31.5 t 66.2 d 125.5 s 108.6 d 143.6 d 140.0 d 15.3 q 49.0 t 62.9 t 99.5 d 171.1 s 170.1 s 21.5 q 20.9 q
22.7 t 25.8 t 31.9 t 62.4 s 53.0 s 203.1 s 89.0 d 47.9 d 50.6 s 52.3 d 33.5 t 65.6 d 125.3 s 108.5 d 143.7 d 140.1 d 14.6 q 48.8 t 64.1 t 104.8 d 171.4 s 170.0 s 21.4 q 21.1 q
a
2.18 1.45 2.38 1.04 4.13 1.97 1.96 2.32 5.87 6.41 7.37 7.42 1.19 3.03 2.37 4.98 4.90 5.25 2.07 2.03 3.38
m m m m s q dd d t dd dd m d dd d d d d s s d
a a a a
3.83 s a a
2.32 5.87 6.40 7.38 7.42 1.00 2.96 2.38 5.70 4.99 5.38 2.07 2.02 4.07
d t dd dd m d dd d d d brd s s d
Overlapped signal.
acetyl derivatives 1b and 3b. Pure acetyl-auropolin (1b) was obtained by Ac2Opyridine treatment of pure auropolin (1a) (Eguren et al., 1981). The hypothesis of a dynamic equilibration between the two hemiacetals 1a and 3a could be ruled out. In fact, auropolin (1a) proved to be stable under mild acidic treatment. Moreover, in other Teucrium species, only one of the two possible hemiacetals was detected, but never both together.
4. Chemotaxonomic significance The distribution of neoclerodanes suggests that T. capitatum is taxonomically close to T. polium subsp. polium as both species contain both capitatin and auropolin; however, T. capitatum also contains many other diterpenoids not present in T. polium subsp. polium. The fact that the diterpenoids occurring in all the taxa indicated as T. polium are
M. Bruno et al. / Biochemical Systematics and Ecology 31 (2003) 1051–1056
1055
distinctly different from those isolated from our authentic T. polium subsp. polium prompts the question of a revision of these identifications.
5. Antifeedant activity A binary choice bioassay using sucrose treated glass-fibre discs (Whatman 2.1 cm diameter) was used to investigate the antifeedant activity of the compounds against final stadium larvae of Spodoptera littoralis (Simmonds et al., 1990) (Table 2). The amounts eaten of the neoclerodane treated (T) and control (C) discs were used to calculate the Feeding Index ((C–T) / (C + T))%. In this index, a positive value indicates an antifeedant. The compounds were tested at a range of concentration from 1 to 1000 ppm to establish whether the compounds gave dose-dependent responses and linear regression was used to calculate the concentration required to give a Feeding Index of 50% (FI50). The Feeding Index at 100 ppm is presented in the results in order to enable comparisons to be made with the activity of other neoclerodanes. The Wilcoxon matched-pairs test was used to analyse the data. A comparison of the antifeedant activity against S. littoralis of the synthetic compound acetyl-auropolin (1b) with the lack of activity of auropolin (1a) shows the importance of the esterification of the hydroxy group at C-20. Compound 4 with a lactone at C-20, although not active at 100 ppm, does elicit antifeedant activity at higher concentration and an FI50 of 230 ppm. Capitatin (2) which lacks a C-7 to C9 bridge showed significant activity at 100 ppm but the activity was not dose-dependent. Previous research on capitatin at 100 ppm showed a Feeding Index (mean (s.e.m.)) of 45 (3.4) and 26 (8.4) against S. littoralis and S. frugiperda, respectively (Simmonds and Blaney, 1992).
Table 2 Effect of compounds 1a, 1b, 2, 4 on the feeding of final stadium larvae of S. littoralis (n = 5–15 per concentration) Compound
1a 1b 2 4
Feeding Indexa at 100 ppm Mean
(SEM)
17.8 57.8∗ 31.0∗ 6.9
7.10 13.64 11.22 19.99
FI50b (ppm)
ndr 90 ndr 230
Feeding Index ((C⫺T) / (C + T))% at 100 ppm (n = 10). Concentration (ppm) required to elicit a 50% Feeding Index: ndr = no linear dose - dependent response. ∗ P ⬍ 0.05 Wilcoxon matched-pairs test. a
b
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M. Bruno et al. / Biochemical Systematics and Ecology 31 (2003) 1051–1056
Acknowledgements The present work was supported by a grant of the Italian Ministry of University and Research (MIUR). We thank Dr Paul Green for technical assistance with the insects, reared under a Plant Health licence from DEFRA, UK.
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